Luminescence microscopy is a typical field of application of light microscopy for examining biological samples. For this purpose, certain dyes (phosphors or fluorophores, as they are called) are used for specific tagging of samples, e.g., of cell parts. As mentioned, the sample is illuminated by excitation radiation and the luminescent light that is excited in this way is acquired by suitable detectors. For this purpose, the light microscope is usually provided with a dichroic beamsplitter combined with blocking filters which split off the fluorescence radiation from the excitation radiation and enable separate observation. This procedure makes it possible to display individual, differently colored cell parts in the light microscope. Naturally, more than one part of a specimen may also be dyed simultaneously with different dyes attaching themselves specifically to different structures of the specimen. This process is known as multiple luminescence. Samples which luminesce, per se, that is, without the addition of dye, can also be measured.
In the present context, and as a general rule, luminescence is used as an umbrella term for phosphorescence and fluorescence and embraces both processes.
Further, it is known to use laser scanning microscopes (LSM) for the examination of samples which shows only those planes situated in the focal plane of the objective in a three-dimensionally illuminated image by means of a confocal detection arrangement (called a confocal LSM in this case) or a nonlinear sample interaction (called multiphoton microscopy). An optical section is acquired and the recording of a plurality of optical sections at different depths of the sample then makes it possible by means of a suitable data processing device to generate a three-dimensional image of the sample which is composed of the different optical sections. Accordingly, laser scanning microscopy is suitable for examining thick specimens.
Naturally, a combination of luminescence microscopy and laser scanning microscopy in which a luminescing sample is imaged at different depth planes by means of a LSM is also used.
In principle, the optical resolution of a light microscope and of a LSM is diffraction-limited by physical laws. Special illumination configurations such as the 4Pi arrangement or arrangements with standing wave fields are known for optimal resolution within these limits. In this way, the resolution can be appreciably improved over a conventional LSM particularly in axial direction. Further, the resolution can be increased by up to a factor of 10 over a diffraction-limited confocal LSM by means of nonlinear depopulation processes.
In recent years, a number of such techniques have been proposed or developed which allow optical microscopy, particularly with LSM, to operate with a resolution beyond the conventional Abbe diffraction barrier [see Y. Garini, B. J. Vermolen and I. T. Young, “From micro to nano: recent advances in high-resolution microscopy”, Curr. Opin. Biotechnol. 16, 3-12 (2005)]. In this connection, there is a basic distinction between nearfield and farfield methods, the latter being especially relevant because of their applicability to three-dimensional imaging in the field of biomedicine.
In conventional fluorescence microscopy with a given numerical aperture (NA) and excitation wavelength, the above-mentioned nonlinear relationship between the intensity of the exciting light and that of the emitted light must be produced in order to break the Abbe barrier of transmissible spatial frequencies in a significant way [see R. Heintzmann, T. M. Jovin and C. Cremer, “Saturated patterned excitation microscopy—a concept for optical resolution improvement”, JOSA A 19, 1599-1609 (2002)]. This is achieved, for example, by means of multiphoton microscopy [see W. Denk, J. H. Strickler and W. W. Webb, “Two-photon fluorescence scanning microscopy, a concept for breaking the diffraction resolution limit”, Science 248, 73-76 (1990)].
Other approaches include the methods of ground state depletion (GSD) [see U.S. Pat. No. 5,866,911 or S. W. Hell and M. Kroug, “Ground-state-depletion fluorescence microscopy: a concept for breaking the diffraction resolution limit”, Appl. Phys. B 60, 495-497 (1995)] or stimulated emission depletion (STED) by Hell et al. [see DE 4416558 C2 S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission; stimulated-emission-depletion fluorescence microscopy”, Opt. Lett. 19, 780-782 (1994); T. A. Klar, E. Engel and S. W. Hell, “Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes”, Phys. Rev. E 64, 066613 (2001); V. Westphal and S. W. Hell, “Nanoscale Resolution in the focal plane of an optical microscope”, PRL 94, 143903 (2005)]. The common principle is based on the use of a distribution of excitation intensity and of saturation intensity in the sample, each of which is structured in such a way that the maximum of the former coincides with an interference minimum of the latter. A saturated excitation of the triplet state (hereinafter: GSD) or a saturated de-excitation of the fluorescing state (hereinafter: STED) makes it possible to deliberately quench the fluorescence of molecules which are not located in the immediate vicinity of the interference minimum. The radiation then proceeds only from the interference minimum. The up-conversion fluorescence depletion technique established by Iketaki et al. functions in a similar way [see T. Watanabe, Y. Iketaki, T. Omatsu, K. Yamamoto, M. Sakai and M. Fujii, “Two-point separation in super-resolution fluorescence microscope based on up-conversion fluorescence depletion technique”, Opt. Exp. 24, 3271-3276 (2003)].
DE 19908883 A1 proposes a direct saturation of the fluorescence transition as a nonlinear process. The enhanced resolution is based on a periodically structured illumination of the sample so that there is a transfer of high object space frequencies in the range of the optical transfer function of the microscope. The transfer can be achieved through costly postprocessing of data by computer.